This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project is designed to evaluate the protective immunogenicity of HIV-1 Envelope (Env) glycoproteins cloned from newly transmitted clade C infections. Based on conserved biological and genetic characteristics we hypothesized that the transmitted Env would induce cross-protective neutralizing antibody responses. We achieved several of our research goals during the reporting period as outlined below: + Developed second system for evaluating matched donor-repicient patient-derived subtype C Envs in a replication-competent subtype C proviral backbone. + Completed the construction of 40 subtype C HIV molecular clones representing the viral variants from 3 matched Donor/Recipient couples from Dr. Susan Allen's Zambian discordant couple cohort. + Performed replication assays on the subtype C HIV molecular clones (above) on U87 cell lines and primary CD4 T cells. The replication kinetics of each molecular clone is being characterized using a mixed variable analysis approach to define the replication phenotype. + Evaluated the replication kinetics of the subtype C HIV molecular clones on primary CD4 T cells, macrophages, CD4 T cell-dendritic cell cocultures and following treatments to increase CCR5 and alpha4-beta7 expression. + Validated the Lac-regulatable dual expression adenovirus vector system that we developed during the previous budget period. We have also characterized gene expression and used electron microscopy to detect HIV VLPs produced from this vector system. + Generated 6 adenovirus vectors that co-express codon-optimized subtype C HIV env and gag genes. + Further developed a collaborative project with Dr. Eric Hunter to test effects of gag CTL escape mutations on replication fitness of subtype C patient-derived HIV-1 using cloning system described above. The significance of this progress is that it provides unique and valuable reagents that are key to understanding the phenotype associated with HIV-1 transmission and the targets for an effective vaccine for protection from HIV-1 infection.